Modification of protein or polypeptide is a naturally occurring phenomenon in vivo, it is primarily that the amino acid residues constituting the polypeptide or protein are modified by some groups, then a series of life activities being regulated in vivo. Typically, the modification may be methylation, acetylation, phosphorylation modification etc. As important lysine methylation modifications, the type of modifications is divided into monomethylation, dimethylation or trimethylation modification (Cheng X, Zhang X. Mutat Res (2007)). How to effectively detect the presence of methylation modification in vivo, especially mono-methylation modification, can provide effective detection of some important diseases with a new method. Because some naturally occurring methylation modifications, such as abnormality of monomethylation modification, may result in some severe diseases such as cancer etc. (Yutaka Kondo et al., Mol Cell Biol. 23(1): 206-215 (2003), Berdasco M et al., Proc Natl Acad Sci, 106: 21830-21835 (2009)).
Lysine methylation modification is a common way of protein modification. Under normal circumstances, detection of protein modification requires antibody specifically identifying the modification, e.g., the protein lysine acetylation modification may be identified by a lysine acetylated pan antibody (Kouzarides, T. Cell, 128, 693-705 (2007), Berger, S. L. Nature, 447, 407-412 (2007)). According to the bonding form of methyl group to lysine, the lysine methylation modification includes three forms of monomethylation, dimethylation and trimethylation. Correspondingly, the development of highly sensitive and highly specific lysine monomethylated, dimethylated and trimethylated antibodies is the premise of detection of the protein lysine methylation modification. (Barski, A. et al., Cell 129, 823-837 (2007); Rea, S. et al., Nature 406, 593-599 (2000)). At present, although successfully developed lysine dimethylated and trimethylated antibodies have been used in detection of lysine dimethylation or trimethylation modification, no successfully developed lysine monomethylated modified antibodies are used in detection of lysine monomethylation modification (Ziqian Liang et al., Proteome Science, Volume 6, (1) 6:2 (2008)). Since the lysine monomethylation modifying group is very small, having a molecular weight of only 15 Da, it is extremely difficult to induce a high immune response, moreover, the organic property of the monomethyl group further weakens immunogenicity of the lysine residues. These intrinsic properties result in great difficulty in antibody development in which the lysine monomethylated group is used as immunogen or the lysine monomethylated modified polypeptide is used as immunogen. Therefore, it is necessary to develop a completely new antibody used in detection of the lysine monomethylation modification.
Mono-, di- or tri-methylation modification may occur on ε-amino group sidechain of the protein lysine residues. In the past few decades, the biological studies on lysine methylation modification (Kme) were mainly focused on core histone. Previous studies showed a key role of such modification in structure and function of chromosome. Such modification state is regulated by two groups of enzymes having opposite catalytic functions, i.e. lysine methyltransferase and lysine demethylase. At present, more than 50 lysine methyltransferases and about 25 lysine demethylases have been found. Histone lysine methylation modification is associated with various diseases, such as cancer. Corresponding, the lysine methylation regulatory enzymes become a series of potential drug targets.
At present the protein post-translational modifications (PTMs) found in histone are all present in non-histone. In the identified lysine methylation regulatory enzymes, there are some non-nuclear targeting enzymes and newly found lysine methylation regulatory enzymes without using histone as substrate, this reveals that the lysine methylation should be widely distributed in non-histone. Identification of the protein substrate is the premise of determination of the protein post-translational modification function, history of the biological study of lysine methylation elaborates the importance of the premise very clearly. After combing our data with other people's data, it is found that that lysine acetylation substrate occurs in three study areas of chromosome structure, transcriptional regulation and metabolism, furthermore these areas overlap mutually in terms of substrate. Although the identification of the substrate for lysine acetylation modification is late, the studies on it indicate that lysine acetylation has synergistic effect in above-described three study areas. Similarly, the identification and quantification of lysine methylation modification group founded some downstream non-histone and signal pathway not being associated with chromosome, this lay a solid foundation for subsequent function study.
However, identification of lysine methylation substrate is not easy. Different from 32P labelling for identifying phosphorylation modification, low radioactivity of 3H or 14C make the radioactive isotope detection method difficult to be realized in the identification of lysine methylation substrate. In methylation, especially monomethylation (Kme1), only one very small structural group is introduced into its substrate, thus difference between the modified lysine group and the unmodified lysine group is slight, only a very small conformation is used for development of affinity antibody. If both of affinity and specificity are taken into account, development of methylation antibody (pan antibody) not being associated with sequence is a great challenge. As a result, because no suitable methylation peptide enrichment method provides peptide for the subsequent mass spectrum analysis, the identification process of the lysine methylation substrate is very slow. As described in phosphorylation and lysine acetylation study, the affinity enrichment is a key step in the global analysis which studies the protein post-translational modification (PTM) (Kim, S. C. et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23, 607-618 (2006)). Because the difference of physicochemical properties between the monomethylation modified lysine and the unmodified lysine, it is very difficult to separate the methylation modified lysine polypeptide by using chemical methods such as the Immobilized Metal Affinity Chromatography (IMAC) for separating the phosphorylated polypeptide (Ficarro, S. B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nature biotechnology 20, 301-305 (2002)). In addition, it is also very difficult to prepare highly specific and high affinity anti-lysine monomethylated antibody. Therefore, it is required to innovate the existing technology based on proteomics so as to identify and analyze the lysine methylation modification. Particularly, it is required to make completely new invention and innovation to the substrate identification and quantitive analysis method for lysine monomethylation modification.